1. Field of the Invention
The necessity and desirability of separating liquid samples such as urine or blood into their respective component parts for examination purposes has long been recognized. It is well known that such a separation can be effected by centrifugation whereby sediments, etc., suspended in the fluid are forced to the bottom of a centrifuge container or test tube, thereby displacing the less dense components to higher levels. In those operations requiring only the sediments (e.g., red blood cell analysis from whole blood, or examination of organized urinary sediments consisting of erythrocytes, leukocytes, epithelial cells, bacteria and casts, as well as unorganized granular or crystalline sediments and the like), typically the supernatant portion is removed and the sediment is resuspended, stained, or otherwise prepared for analysis.
The need for standardized and reproducible techniques for sample preparation and examination is well established. The initial sample volume and the sediment sample volume must be controlled, as well as operator techniques overall and especially in the removal of the supernatant fluid after centrifugation, so that the sediments are conserved and the results are accurate and reproducible. The same requirements obtain for the separation of liquid phase systems.
2. Prior Art
Several past efforts to fill this need have generally provided means for placing a physical barrier into a test tube containing a sample between the sediment and the supernatant before or after the centrifugation. The barrier would typically be designed to remain in place while the supernatant is decanted, aspirated or otherwise poured off. Subsequently, the physical barrier would need to be removed to provide access to the sediment remaining in the test tube for purposes of resuspension, introduction of stain, or removal for examination.
The Parker patent (U.S. Pat. No. 4,022,576) illustrates such a device to be inserted after centrifugation. It provides an insert member which is hollow, closed at its upper end and open at the end to be inserted into the tube. The insert member is further provided with a bead of appropriate size which wedges into a test tube at a point of reduced cross section, and provides a fluid-tight physical barrier at a predetermined distance from the bottom of the tube. The liquids above this barrier are decanted, and then the insert member is removed to allow for the addition of stain to the fraction remaining in the tube, or alternatively, some of the sediment sample remaining in the bottom portion of the test tube may be withdrawn into the hollow insert member, and subsequently the entire insert member is removed from the tube in order to transport the sample contained therein.
The Parker patent has several disadvantages. The primary problem is that proper placement of the insert member in the test tube requires it to be wedged therein, thus it introduces elements of inaccuracy and nonreproducibility, since the firmness of the operator's wedging technique can vary the volume trapped in the test tube below the wedge and thus improperly concentrate or dilute the sediment. Further, due to the sealing engagement provided by the wedge, the insert member must be forcibly released from the test tube; this step is time-consuming and requires pressure to be exerted on the insert member, which increases the risk of losing some of the sample which has been drawn up into the insert member for transfer. Additional time and efficiency is wasted if the sample is to be stained for examination while remaining in the tube, because the insert member must first be entirely removed to provide an opening for the introduction of the stain, and then subsequently reintroduced to transfer the sample elsewhere from the test tube.
An alternative approach to this problem is shown in R. K. Bernstein et al., (U.S. Pat. No. 3,481,712) which utilizes a system which confines the sediments in a liquid sample to the bottom of a centrifuge tube without providing a physical barrier between the supernatant and the sediment phases. This system utilizes a reservoir at the lower end of the centrifuge tube of relatively reduced cross section, further especially adapted to retain a predetermined volume within by the means of surface tension and atmospheric pressure.
While it is advantageous to be thus free from the necessity of removing any insert means to stain or remove the sediment, the lack of any physical restraining means causes a problem at certain standard experimental volumes. Using accepted and standard procedures well known in the art, generally about 10-15 ml of a liquid sample are centrifuged for about five minutes at 400.times. g (i.e., 400 times the gravitational acceleration force). (See, e.g. Parker, Bernstein). The solids contained in the liquid, a urine specimen for example, are thereby normally concentrated in the lower 1 ml portion of a centrifuge tube. A preferred embodiment of the Bernstein invention (col. 3, lines 62-75), in contrast, operates at smaller volumes, utilizing 4.1 ml of whole sample and obtaining 0.15 ml of sediment.
In order to obtain the larger standard 1 ml amounts of sediment typically required for analytical purposes, more of a whole sample would be needed, necessitating a larger container and also an enlargement of the sediment reservoir (perhaps by two-thirds) compared to the preferred embodiment. However this would be disadvantageous, as increasing the reservoir in depth or in its cross section would change the surface tension in the apparatus which governs the sediment collection, and thus render experimental results less readily reproducible.
There has long been a need in the field of laboratory analysis for a method and apparatus for accurate, reproducible preparation of liquid-phase specimens and the like, for further chemical and microscopic analysis, which method and apparatus further provides access to two liquid phases without requiring time-consuming and inefficient disruption of the apparatus.
The present invention provides such an enhanced method and apparatus for sample preparation to achieve accurate and reproducible microscopic or chemical examination.